Ribosome biogenesis in eukaryotes requires the participation of several transactivation factors that are involved in the modification, assembly, transport and quality control of the ribosomal subunits . One of these factors is the Large subunit GTPase 1 (Lsg1), a protein that acts as the release factor for the export adaptor named Nonsense-mediated mRNA decay 3 protein (Nmd3) and facilitates the incorporation of the last structural protein uL16 into the 60S subunit . Here, we characterised the recombinant yeast Lsg1 and studied its catalysis and binding properties for guanine nucleotides . We described the interaction of Lsg1 with guanine nucleotides alone and in the presence of the complex Nmd3•60S using fluorescence spectroscopy . Lsg1 has a greater affinity for GTP than for GDP suggesting that in the cell cytoplasm it exists mainly bound to the former . In the presence of 60S subunits loaded with Nmd3, the affinity of Lsg1 for both nucleotides increases but to a larger extent towards GTP . From this observation together with the excess of GTP present in the cytoplasm of exponentially growing cells over that of GDP, we can infer that the pre-ribosomal particle composed by Nmd3•60S acts as a GTP Stabilising Factor for Lsg1 . Additionally, Lsg1 undergoes different conformational changes depending on its binding partner or the guanine nucleotides it interacts with . Steady-state kinetic analysis of free Lsg1 indicated slow GTP hydrolysis with values of k 1 min and K of 34 μM.